Abstract
Loss-of-function studies have demonstrated the essential role of Notch in definitive embryonic mouse hematopoiesis. We report here the consequences of Notch gain-of-function in mouse embryo hematopoiesis, achieved by constitutive expression of Notch1 intracellular domain (N1ICD) in angiopoietin receptor tyrosine kinase receptor-2 (Tie2)-derived enhanced green fluorescence protein (EGFP+) hematovascular progenitors. At E9.5, N1ICD expression led to the absence of the dorsal aorta hematopoietic clusters and of definitive hematopoiesis. The EGFP+ transient multipotent progenitors, purified from E9.5 to 10.5 Tie2-Cre;N1ICD yolk sac (YS) cells, had strongly reduced hematopoietic potential, whereas they had increased numbers of hemogenic endothelial cells. Late erythroid cell differentiation stages and mature myeloid cells (Gr1+, MPO+) were also strongly decreased. In contrast, EGFP+ erythro-myeloid progenitors, immature and intermediate differentiation stages of YS erythroid and myeloid cell lineages, were expanded. Tie2-Cre;N1ICD YS had reduced numbers of CD41++ megakaryocytes, and these produced reduced below-normal numbers of immature colonies in vitro and their terminal differentiation was blocked. Cells from Tie2-Cre;N1ICD YS had a higher proliferation rate and lower apoptosis than wild-type (WT) YS cells. Quantitative gene expression analysis of FACS-purified EGFP+ YS progenitors revealed upregulation of Notch1-related genes and alterations in genes involved in hematopoietic differentiation. These results represent the first in vivo evidence of a role for Notch signaling in YS transient definitive hematopoiesis. Our results show that constitutive Notch1 activation in Tie2+ cells hampers YS hematopoiesis of E9.5 embryos and demonstrate that Notch signaling regulates this process by balancing the proliferation and differentiation dynamics of lineage-restricted intermediate progenitors.
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Abbreviations
- 7-AAD:
-
7-aminoactinomycin D
- AGM:
-
aorta-gonads-mesonephros
- BM:
-
bone marrow
- DAPI:
-
4′,6-diamidino-2-phenylindole
- EdU:
-
5-ethinyl-2′-deoxyuridine
- EGFP:
-
enhanced green fluorescence protein
- FSC:
-
forward scatter
- HSC:
-
hematopoietic stem cells
- MFI:
-
mean fluorescence intensity
- MK:
-
megakaryocyte
- mAbs:
-
monoclonal antibodies
- N1ICD:
-
Notch1 intracellular domain
- P-Sp:
-
para-aortic splanchnopleura region
- PI:
-
propidium iodide
- qRT-PCR:
-
quantitative real-time PCR
- SSC:
-
side scatter
- Tie2:
-
tyrosine kinase receptor-2
- vWF:
-
von Willebrand factor
- WT:
-
wild type
- YS:
-
yolk sac
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Acknowledgements
We are grateful to B Palacios, P Martínez and C Ruiz for husbandry, Dr. M Alía and C Prado for their technical cytometry assistance and Dr. N Serrano and Dr. B de Andrés for helpful discussions. We thank S Bartlett (CNIC) for English editing. IC is supported by a fellowship from the Ministerio de Economía y Competitividad (MINECO). PMR has been supported by a PhD fellowship (FPU program, MINECO) and by the Fundación Leticia Castillejo. This work was funded by grants SAF2007-65265, SAF2009-12596, SAF2012-33916, and ISCIII08/0685 (MINECO) and SAL-0304-2006 (Madrid Regional Government) to MLG; SAF2007-62445 and SAF2010-17555, RD12/0019/0003 and RD12/0042/0005 (MINECO) to JLdlP. The CNIC is supported by the MINECO and the Pro-CNIC Foundation.
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MARM, MLG and JLP designed research; IC, PMR, LL and MSA performed research; IC and MLG contributed analytical tools; IC, PMR, MARM, MLG and JLP analyzed and interpreted data; IC and MLG performed statistical analysis; JLP, MLG, IC and PMR wrote the manuscript.
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Cortegano, I., Melgar-Rojas, P., Luna-Zurita, L. et al. Notch1 regulates progenitor cell proliferation and differentiation during mouse yolk sac hematopoiesis. Cell Death Differ 21, 1081–1094 (2014). https://doi.org/10.1038/cdd.2014.27
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DOI: https://doi.org/10.1038/cdd.2014.27
